Progesterone's role in deep infiltrating endometriosis: Progesterone receptor and estrogen metabolism enzymes expression and physiological changes in primary endometrial stromal cell culture

Highlights

• Deep infiltrating endometriosis stromal down expresses progesterone receptor.

• CYP19 are main character in deep infiltrating endometriosis cells estrogen metabolism disruption.

• HSD17B2 is overexpressed in deep infiltrating endometriosis primary cells.

• Deep infiltrating endometriosis primary cells do not respond to progesterone stimulus in vitro.

Abstract

To study progesterone signaling activation, we measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE) using a cytosensor microphysiometer and assessed progesterone receptor (PR) and estrogen metabolism enzymes mRNA expression in cultured endometrial cells from women with deep infiltrating endometriosis and healthy controls using real-time quantitative PCR. This study was conducted at a University hospital and included patients with and without deep infiltrating endometriosis (DIE). Primary endometrial stromal cells (ECs) from women with DIE and controls were treated with 17β-estradiol and progesterone prior to microphysiometer measurements and qPCR evaluations. Decreased progesterone responsiveness and decreased total nuclear PR and HSD17B1 mRNA expression were observed in cultured ECs from women with deep infiltrating endometriosis relative to those from control samples before and after hormone treatment. These cells also showed increased 17β-hydroxysteroid dehydrogenases types 2 (HSD17B2) relative to control group and increased expression of aromatase (CYP19) after exposure to progesterone. These physiological and expression patterns observed in ECs cultures from women with DIE reinforces previous findings in the literature supporting the progesterone resistance hypothesis in the pathogenesis of endometriosis.

Introduction

Endometriosis is a chronic inflammatory disease characterized by the implantation and growth of endometrial tissue outside of the uterus. This disease affects 10% of women of reproductive age and is significantly associated with infertility, chronic pain, and morbidity. Deep infiltrating endometriosis (DIE), which occurs in approximately 1% of women of reproductive age is characterized by the penetration of endometrial-like tissue >5 mm under the peritoneal surface (Koninckx and Martin, 1994) and represents the most severe form of endometriosis causing severe pain in the vast majority of affected women. It is characterized by the presence of rectovaginal nodules and disease in the uterosacral ligaments, rectum, rectovaginal septum, vaginal and bladder (Giudice, 2010; Berlanda et al., 2017; Johnson et al., 2017).

Environmental, immune, and hormonal factors in particular are associated with the etiology of the endometriosis. Several studies pointed that women with the disease unveil several abnormalities in the eutopic endometrium, raising questions about whether the uterine mucosa is involved in the pathogenesis of the disease, a hypothesis known as the endometrial theory (Vinatier et al., 2000; Bulun et al., 2006).

Moreover, several local hormonal abnormalities have been observed in the topic endometrium of women with the disease. One interesting aspect of endometriosis is its dependence on estrogen for growth, similar to that seen in the eutopic endometrium, but with a lack of a consistent response to progesterone in some women with deep infiltrating disease (Abdelsamie et al., 2017). Substantial molecular differences in the progesterone response of normal endometrium and ectopic tissues from women with endometriosis have been observed. According to recent reports, the resistance of endometrial tissue to progesterone, which has been shown in both laboratory and clinical observations, is partially explained by abnormalities in the distribution of progesterone receptor (PR) isoforms and the dysregulation of progesterone target genes (Barragan et al., 2016; Kao et al., 2003; OSTEEN et al., 2002; Zeitoun et al., 1998).

The expression of the enzymes related to the estrogen metabolism have been also related to the pathogenesis of the disease and have controversial results among the literature (Rižner, 2009). It is well known that the metabolic pathways in endometriosis are imbalanced in the sense of augmented estradiol (E2) biosynthesis and lower E2 inactivation leading to increased proliferation of the endometrioctic tissues (Giudice and Kao, 2004). The main responsible for this imbalance seems to be the down or absent expression of 17β-hydroxysteroid dehydrogenases types 2 (HSD17B2) even with progesterone stimulus (Zeitoun et al., 1998; Dassen et al., 2007; Šmuc et al., 2009; Attar and Bulun, 2006). Although some studies suggest HSD17B2 is higher in DIE and ovarian endometriosis both mRNA and protein (Matsuzaki et al., 2004, 2006; Carneiro et al., 2007). Osiński et al. found a non-statistically significant increasing trend of HSD17B2 transcript levels in the eutopic endometrium during the folicular phase (Osiński et al., 2018). Also, other enzymes such as aromatase (CYP19), 17β-Hydroxysteroid dehydrogenase types 1 (HSD17B1) and 4 (HSD17B4), steroids sulfatase (STS) and steroidogenic acute regulatory protein (StAR) were also reported to be involved in the impaired estrogenic metabolism in endometriosis (Rižner, 2009; Matsuzaki et al., 2004, 2006; Kitawaki et al., 2003).

Based on previous evidence of endometrial resistance to progesterone and its involvement in the etiology and development of endometriosis, we aimed to verify, in vitro, whether this phenomenon is also present in samples from women with deep infiltrating endometriosis. For that, we assessed progesterone receptor (PR), aromatase – CYP19, 17β-Hydroxysteroid dehydrogenase types 1, 2 and 4 – HSD17B1, HSD17B2 and HSD17B4; steroids sulfatase – STS and steroidogenic acute regulatory protein – StAR mRNA expression in cultured endometrial cells from women with deep infiltrating endometriosis and healthy controls using real-time quantitative PCR and measured changes in extracellular pH as a reflection of Na+/H+ exchange (NHE) using a cytosensor microphysiometer after 17β-estradiol and progesterone stimulation.